Corticotropin-releasing factor (CRF) is a major hypothalamic hormone that regulates the functions of the hypothalamic-pituitary-adrenal (HPA) axis. CRF acts directly upon the specific receptor(s) located on the plasma membrane of pituitary corticotrophs. Additionally, specific CRF binding-sites were reported in neuronal, immune, mucosal, and testicular tissues. The CRF receptor is a membrane glycoprotein that is regulated by CRF, glucocorticoids, stress and other factors, such as vasopressin. CRF binds to these receptors and activates adenylate cyclase, leading to the accumulation of cAMP within the cells. cAMP binds to the regulatory subunit of the cAMP-dependent protein kinase (PKA), leading to dissociation of its catalytic subunits. The catalytic subunit of PKA phosphorylates specific regulatory proteins which bind to promoter sequences and regulate gene transcription. The major goal of this project is to understand the molecular mechanisms of activation of the CRF receptor by CRF; and stimulation of the intracellular signalling cascade by the CRF receptor. In Specific Aim I, we will clone, sequence and express the sheep CRF receptor cDNA. The functions of the cloned receptor will be analyzed in a transient expression system. In Specific Aim II, we will clone the human cDNA and the human gene encoding the CRF receptor. This will be particularly important to study conditions associated with CRF receptor dysfunctions and to determine whether one or multiple CRF receptor subtypes exist in the human genome. In Specific Aim Ill we will analyze the structural requirement for CRF binding and determine the binding site by specific site-directed mutagenesis and mutation/deletion analysis of the cloned receptor cDNA. This will guide the synthesis of receptor fragments that function as physiologic antagonists and may have clinical application. In Specific Aim IV we will characterize the receptor domains that are important for activation of intracellular effectors. This will lead to the characterization of receptors with novel biologic properties. In Specific Aim V we will study the molecular mechanisms of CRF receptor regulation. This aspect is very important since downregulation of the CRF receptor is an important mechanism by which the CRF effects are switched off. Achieving the goals of this research will provide the basic knowledge to understand the role of the CRF in the physiology of the HPA axis.